Method for reproducing conifers by somatic embryogenesis using galactose containing compounds as a carbon and energy source

ABSTRACT

A method for reproducing conifers by somatic embryogenesis is disclosed. A galactose-containing compound is used as a carbon source for an embryogenic culture during at least one of the steps of induction, proliferation, and prematuration.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional PatentApplication No. 60/442,107, filed Jan. 23, 2003, entitled “A METHOD FORREPRODUCING CONIFERS BY SOMATIC EMBRYOGENESIS USING GALACTOSE CONTAININGCOMPOUNDS AS A CARBON AND ENERGY SOURCE.”

BACKGROUND OF THE INVENTION

[0002] Somatic embryogenesis offers the potential to clonally producelarge numbers of plants of many species at low cost. Somatic embryosdevelop without the surrounding nutritive tissues and protective seedcoat found with zygotic embryos, so research has been directed tocausing somatic embryos to functionally mimic seeds with regard toefficient storage and handling qualities. The development of techniquesfor somatic embryogenesis in conifers has greatly improved the abilityto culture conifer tissues in vitro and now offers the means to clonallypropagate commercially valuable conifers. However, it is necessary tofurther reduce production costs to make somatic embryogenesis affordableto industry. Thus, there is a need in the technology for improvement ofthe efficiency of embryo production and of the quality and vigour ofplants resulting from somatic embryos from all species of conifers.

BRIEF SUMMARY OF THE INVENTION

[0003] A method for reproducing conifers by somatic embryogenesis isdisclosed wherein a galactose-containing compound is used as a carbonsource for an embryogenic culture during at least one of the steps ofinduction, proliferation, and prematuration.

BRIEF DESCRIPTION OF THE FIGURES

[0004]FIG. 1 represents the ability of loblolly pine lines grown on TXmaintenance medium to hydrolyze sucrose over time.

[0005]FIG. 2 represents the growth rate of loblolly pine lines grown onTX maintenance medium.

[0006]FIG. 3 represents the growth rate of loblolly pine lines grown onprematuration medium I containing lactose.

[0007]FIG. 4 represents the growth rate of loblolly pine lines grown onprematuration medium II containing lactose.

[0008]FIG. 5 represents the ability of loblolly pine lines grown onprematuration medium I and utilise lactose by breaking it down toproduce glucose and galactose.

[0009]FIG. 6 represents the ability of loblolly pine lines grown onprematuration medium II to utilise lactose by breaking it down toproduce glucose and galactose.

DETAILED DESCRIPTION

[0010] The following definitions are those consistent with the usage ofterms in the present specification.

[0011] Abscisic Acid (ABA) A plant growth regulator in the group ofstress hormones.

[0012] Auxin A plant growth regulator which may be natural or synthetic.The main physiological effect of auxin is to stimulate cell elongation.Examples are indole acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid(2,4-D).

[0013] Clone When used in the context of plant propagation, the termrefers to a collection of individuals having the same geneticconstitution (genotype), and is produced from a culture that arises froman individual explant.

[0014] Conversion The ability of a somatic embryo to germinate (eitherin vitro or ex vitro) and subsequently develop into an established,autotrophic plant with root and needles.

[0015] Cytokinin A natural or synthetic plant growth regulator such asbenzyl adenine (BA). The main physiological effect of cytokinin is tostimulate meristematic cell division.

[0016] Desiccation The drying of an embryo by any means to a moisturecontent less than that of the original hydrated embryo. Desiccationprocesses may include (a) mild desiccation, which encompasses moisturecontents in the 36-55% moisture content range, and (b) severedesiccation, which occurs at moisture contents less than 36%, usually inthe range of about 5-30%. A severely desiccated viable embryo is able tosurvive freezing, and after rehydration, is able to successfullycomplete the germination process and convert to a normal autotrophicplant.

[0017] Embryogenic culture A plant cell or tissue culture capable offorming somatic embryos and regenerating plants via somaticembryogenesis.

[0018] Explant Organs, tissues or cells derived from a plant andcultured in vitro for the purposes of starting a plant cell or tissueculture.

[0019] Galactose A hexose of the formula CH₂OH.(CHOH)₄CHO. It is presentin certain gums and seaweeds as a polysaccharide galactan and as anormal constituent of milk.

[0020] Lactose Lactose is a disaccharide (C₁₂H₂₂O₁₁). It yieldsD-glucose and D-galactose on hydrolysis, which is catalysed by lactase.

[0021] Line This is another term for “clone”.

[0022] Mature embryo A mature embryo is one which is capable ofgermination to yield a seedling with shoot and root, given the necessaryenvironmental conditions (temperature, light, water, nutrients, etc.).The term implies that the embryo has undergone development throughvarious developmental stages and has reached a size and stage suitablefor germination. The embryo contains storage proteins, lipids, and ifprovided with suitable maturation conditions (e.g. ABA and waterstress), will be desiccation tolerant, so may be desiccated prior togermination.

[0023] Megagametophyte A haploid nutritive tissue of gymnosperm seed, ofmaternal origin, within which the gymnosperm zygotic embryos develop.

[0024] Moisture content The amount of water present in an embryo. Thisis generally measured by weighing an embryo before and after oven drying(FW and DW, respectively). The preferred manner of expression ispercentage weight of water based on the original weight of the embryo,so that the values are always less than 100. MC=((FW−DW)/FW)×100%

[0025] Prematuration The step following the proliferation step and priorto the maturation step, usually involving a gradual reduction in theconcentrations of one or more of the hormones auxin and cytokinin and/ora change in water stress and the addition of ABA.

[0026] Proliferation The steps following induction prior to maturation,in which embryogenic cultures divide and grow but do not develop intomature embryo stages. The proliferation step may also be referred to asthe maintenance step.

[0027] Nutrients The inorganic micro- and macro-minerals, vitamins,hormones, organic supplements, and carbohydrates (or any one or more ofthem) necessary for culture growth and somatic embryo germination.

[0028] Somatic embryo A plant embryo formed in vitro from vegetative(somatic) cells by mitotic division of cells. Early stage somaticembryos are morphologically similar to immature zygotic embryos, andcomprise a region of embryonal cells subtended by suspensor cells.

[0029] Somatic Embryogenesis A process of initiation and development ofsomatic embryos in vitro from somatic cells and tissues.

[0030] Water potential The total water potential to which an organism issubjected in a water-containing matrix. This is a sum of (1) osmotic(solute) potential, (2) gravitational potential resulting from thevertical position of the water, and (3) a suction component (capillaryor matric potential).

[0031] Water Stressing The reduction of water potential to which anembryogenic tissue or somatic embryo is controlled during maturation bycontrolling the environment of the tissue or embryo in order to modifythe progress of maturation.

[0032] Zygotic Embryo An embryo derived from the sexual fusion ofgametic cells produced by meiosis.

[0033] Somatic embryogenesis in plants is a multistep process consistingof induction, proliferation, maturation (embryo development), andgermination and requires that specific culture conditions, includingnutrient media compositions, are provided for each step of the multistepprocess. Thus, in general for most conifers an auxin and cytokinin and alow osmoticum are required in media for induction and proliferation ofembryogenic tissues. For further embryo development it is oftenbeneficial to increase the osmotic concentration, and to replace theauxin and cytokinin with abscisic acid (ABA). The effects of differentcarbohydrates on the different steps of somatic embryogenesis are notclearly understood. For conifers the optimal carbohydrate has thus farbeen found to be either sucrose or maltose (Iraqi and Tremblay 2001 a,Iraqi and Temblay 2001b, Ramarosandratana et al, 2001).

[0034] Water stress plays an important role in maintaining embryos in amaturation state, and low water content rather than ABA may preventprecocious germination during later stages of development. This isimportant because precocious germination of embryos during developmentin seeds would be lethal during desiccation. A conventional way to waterstress plant cells grown in vitro is to increase the osmoticconcentration of the culture medium through the use of plasmolysingosmotica. For example, increased concentrations of plasmolysing osmoticasuch as sucrose or mannitol have been used to promote somatic embryomaturation of many plant species.

[0035] Sucrose or maltose are considered to be the most suitable carbonsources for conifers, depending upon the species, for most stages ofculture. Lactose has been used as an osmoticum with maltose or sucrosesupplements as the carbon source during the conifer maturation step.Lactose or galactose has not been described as a suitable energy sourcefor the induction or maintenance of the immature embryo stages. Thenewly-discovered fact that embryos of some conifer species can hydrolyzelactose and/or metabolize galactose at certain developmental stageswithout galactose toxicity, and that this results in improved culturesis an unexpected beneficial result.

[0036] Here, a compound containing galactose as a carbon and energysource in the media is used during early conifer embryogenesis,specifically sugars containing galactose or galactose subunits. The mostsuitable sugars are galactose or lactose, the choice of which depends onthe conifer species being cultured.

[0037] The method is especially well suited to culturing conifers of thefamily Pinaceae, especially those species including Pinus taeda(loblolly pine), Pinus radiata (radiata pine) and Pseudotsuga menziesii(Douglas fir). The method produces higher induction frequencies.

[0038] The method promotes differentiation of immature embryos inproliferation cultures. These embryos when subsequently transferred todevelopment (maturation) medium show reduced growth of suspensor tissue,improved embryo quality and improved yields of mature embryos. Thisresults in higher numbers of genotypes that can be successfully culturedand cryostored, and results in a higher number of seedlings produced atlower costs than those of prior known methods.

[0039] Diploid cultures of conifers are most readily initiated fromzygotic embryos, which are genetically dissimilar from each other. Lowosmotic conditions are beneficial for induction from conifers. The sugarmost often used for induction is sucrose at about 1% (w/v)concentration. However, the use of galactose-containing compounds, suchas galactose and lactose at the induction stage, is now shown to lead toimproved induction success. The most suitable concentration may be lessthan about 6%, and, more particularly, may be less than about 2%, andeven more particularly, between about 1% and about 2%, alone or withadditional carbon sources. Cytokinin is important to most species, andis usually included with an auxin at concentrations of 5 and 10 μM,respectively.

[0040] Exceptions to the above plant growth regulator (PGR) requirementsare Abies spp, which are different from other conifers, and requirecytokinin as the sole PGR for induction as well as proliferation.

[0041] Genotype specificity for embryogenic induction occurs inconifers. Also, the induction of somatic embryogenesis is under strongadditive genetic control. For conifers, immature zygotic embryos yieldsomatic embryos more readily than mature zygotic embryos, which aregenerally more responsive than explants from young seedlings.Cryopreservation of immature somatic embryos in liquid nitrogen isroutine for long-term preservation and is used to preserve genotypeswhile extended field tests are carried out. Following the field trialselite genotypes are then removed from cryogenic storage and bulked up inliquid suspensions for mass propagation. Methods of this invention leadto greater numbers of cryopreserved lines following induction.

[0042] Embryogenic cultures of conifers are usually maintained on amedium similar to the induction medium, which typically includes anauxin, a cytokinin and a low concentration of sugar. The method ofmaintenance (or proliferation) depends upon the intended use of theculture. A common way to maintain cultures is on semi-solidified mediumin Petri dishes. These stationary cultures are sub-cultured every 2-4weeks to prevent browning and death. Liquid culture is more suitable formaintaining conifer cultures in a rapidly proliferating state for largescale propagation.

[0043] Pine embryogenic tissue, when grown on abscisic acid (ABA) in thepresence of sucrose, undergoes a disorganised growth phase, prior toorganised growth. Such cultures do not readily undergo furtherdevelopment with ABA and a low osmotic concentration, perhaps becausethey are too juvenile to respond to ABA. Replacing the sucrose withlactose or galactose at the proliferation step has the benefit ofreducing disorganised growth, and leads to the production ofwell-organised immature embryos with enlarged embryonal regions. Thiseffect is more pronounced than with maltose. When transferred tomaturation medium these lactose/galactose embryos have a greaterpropensity to develop to mature embryo stages than those grown on othersugars, including maltose, and disorganised suspensor tissueproliferation is inhibited. The result is that mature embryos areproduced in higher yields than with other known methods, and the matureembryos are of greater uniformity. They are also of better quality andthus are vigorous during subsequent germination. All of the latter leadto greater numbers of plants recovered at the end of the process.

[0044] In order to encourage the production of mature developmentalstages of conifer somatic embryos, immature somatic embryos must betransferred from a medium containing hormones to stimulate proliferationto an environment containing ABA and ideally a raised osmoticconcentration. A gradual transition to these growth conditions is oftenbeneficial. Thus, prior to ABA treatments, immature somatic embryos maybe transferred to a prematuration medium containing no, or reduced,plant growth regulators. Charcoal may be beneficial.

[0045] Following the maturation step it is often desirable to desiccatethe somatic embryos. A moisture content of less than 55% is beneficialto producing high storage reserves and inducing desiccation tolerance.In addition to promoting germination, desiccation reduces productioncosts by providing a means of storing somatic embryos. For optimalefficiency, mature somatic embryos can be produced continuously yearround, then stored and pooled with somatic embryos from subsequentproduction runs. They can then be germinated synchronously to provideplants of uniform age and size for planting during a suitable growingseason.

[0046] Post-germinative growth of conifer somatic embryos occurs withoutthe benefit of the haploid megagametophyte, which is a major organ forstorage of both lipids and proteins within the conifer seed. Conifersomatic embryos therefore require nutrients, usually in the form ofPGR-free media supplied at half strength and containing 1-3% sucrose forfurther growth into autotrophic plants.

[0047] The process of the present invention is not limited to any singlebasal culture medium. Any well known medium or modification may be used,however we have found the formulation below to work well for manyconifers as described in the following sections. TABLE A TX medium basalsalts Amount per liter Basal salts Medium, mg Major KNO₃ 950.00 KH₂PO₄170.00 MgSO₄.7H₂O 925.00 CaCl₂.2H₂O 211.00 Minor KI 4.15 H₃BO₃ 31.0000ZnSO₄.7H₂O 43.0000 MnSO₄.H₂O 21.0000 Na₂MoO₄.2H₂O 1.5000 CuSO₄.5H₂O0.5000 CoCl₂.6H₂O 0.1300 Iron FeSO₄.7H₂O 27.8 Na₂EDTA 37.2 VitaminsThiamine-HCl 0.1000 Pyridoxine-HCl 0.1000 Nicotinic acid 0.5000Myo-Inositol 100

EXAMPLES

[0048] Examples Assessing Loblolly Pine Somatic Embryogenesis

Example 1 Effect of Carbon Source on Induction of Loblolly Pine

[0049] Female gametophytes containing the immature zygotic embryos wereused as explants. The cones were surface sterilized in 10% bleachsolution for 15 min. They were rinsed 3 times with sterile water, airdried and stored in polyethylene bags at 4° C. until used. Seeds weresterilized in 3% hydrogen peroxide containing 0.1% Tween 20 and rinsedin sterile deionized water. Megagametophytes containing the immatureembryos were plated on TX medium (Table A) supplemented with 2.2 mg/l2,4-D, 1 mg/l BA and 0.3% Phytagel and containing either 1% sucrose, or1.5% lactose and 0.025% glucose.

[0050] Cultures were maintained on the two media by subculturing theembryogenic tissue every 10 to 14 days to fresh media. All cultures wereincubated at 23° C. in the dark. The results of the induction arepresented in Table 1. TABLE 1 Effect of carbon source on Loblolly pineinduction success Treatment % induction TX sucrose 1.46 TX lactose 6.85

[0051] Induction success was greater than three times higher when mediumcontaining lactose was used.

Example 2 Effect of Carbon Source on Solid Maintenance of Loblolly Pine

[0052] Tissue induced on sucrose from Example 1 was further maintainedon the semi-solid TX medium containing 1% sucrose, and tissue induced onlactose was maintained on semi-solid TX medium containing 1.5% lactoseand 0.025% glucose. The tissue morphology of the lines induced onlactose was different from the ones induced on sucrose. Tissue inducedon lactose-containing medium consisted of more early-stage immatureembryos and a limited amount of suspensor type of surrounding tissue.Predominantly suspensor type tissue and very few embryos were observedin the tissue induced on sucrose. In order to further increase theproliferation rate, 0.5% sucrose was added to the TX 1.5%lactose-containing medium in place of 0.025% glucose. This mediumprovided a reasonable bulk-up rate, and growth of embryos was moresynchronized than in the 1% sucrose medium.

Example 3 Effect of Carbon Source on Liquid Maintenance

[0053] Suspension cultures of loblolly lines LP1, LP2, LP3, LP4 and LP5were started with 2 g of tissue introduced to 50 ml of TX liquid mediumcontaining different sugars—1% sucrose, 1.5% maltose, or 1.5% lactose.These cultures were maintained in the same liquid media for 2 weeks byweekly transfers to fresh maintenance medium containing the abovesugars. Then, 2 g of tissue produced in each maintenance medium wastransferred to prematuration media I, containing the same carbon sourceas the maintenance media. After one week, 1.5 g of tissue from eachprematuration medium I was transferred to prematuration media IIcontaining the same sugars. After the 4 week period in liquid media,early stage somatic embryos were collected and plated on ½ LV maturationmedium containing 120 μM ABA and 6.0% sucrose and 1% Phytagel. Eight toten weeks later, mature somatic embryos were counted and treatments wereevaluated based on the number of somatic embryos per gram fresh weight(SE/g FW). Table 2 summarises the tissue yield at the end of the 4-weekperiod and the number of mature somatic embryos produced for each line.TABLE 2 Effect of carbon source on growth rate and yield of somaticembryos. Average amount of tissue produced after 4 weeks in Carbonsource liquid (FW g) # SE/g FW ± sd   1% Sucrose 592.06 126 ± 20 1.5%Maltose 204.45 209 ± 28 1.5% Lactose 18.63 393 ± 10

[0054] The highest growth rate was observed when sucrose was used forthe entire 4-week liquid period. The lowest amount of tissue wasobtained when lactose was used as sole carbon source. However, tissuecultured in lactose-containing media produced the highest number of SE/gFW. This significantly reduces production costs, as about 60% lessmaturation cultures are required to produce a target number of embryos.

Example 4 Effect of Lactose During the Prematuration Steps

[0055] Prematuration for loblolly pine may be performed in two steps:

[0056] 1. Prematuration I requires reduction of hormones either byreducing the auxin and cytokinin concentrations to optimally about{fraction (1/20)} of the concentration of proliferation step, or byadding charcoal at about 0.01 to 1%, or a combination of both.

[0057] 2. Prematuration II follows prematuration I and requires anaddition of ABA at concentrations of about 5 to 120 μM, preferably 90 μMand no auxin and cytokinins.

[0058] In order to further understand the effect of lactose on somaticembryo development, liquid maintenance cultures were started in TXmedium containing 1% sucrose as in Example 3. Three lines were used inthis experiment, LP2, LP3 and LP4. After the proliferation step, 2 g ofthe tissue was transferred for one week to prematuration medium Icontaining different concentrations of lactose—1.5%, 3% and 6%. Afterthat, 1.5 g of tissue produced in the lactose-containing media wasfurther cultured for a week in prematuration medium II containing thesame amount of lactose. The summary of the results is presented inTable. 3. TABLE 3 Effect of lactose at the prematuration step. Meangrams FW at the end of Carbon source prematuration stage Mean # SE/g FW± sd 1% Sucrose to 1.5% lactose 121.90 106 ± 9 1% Sucrose to 3% lactose216.47 209 ± 7 1% Sucrose to 6% lactose 149.13  169 ± 10

[0059] The highest numbers of somatic embryos were obtained when 3%lactose was used for the last two weeks of the liquid prematurationstep.

Example 5 Carbohydrate Metabolism

[0060] In order to understand the effect of sucrose and lactose onsomatic embryo development, all media required for the liquid stage(maintenance, prematuration I and II) were analysed for sucrose,glucose, lactose, and galactose. Two lines were used in this experiment:LP3 and LP6.

[0061] Both lines completely hydrolysed sucrose at day 5 during thefirst week of culture in TX maintenance medium. A similar pattern in theability of the lines to hydrolyse sucrose was observed aftertransferring the tissue to the same medium for an additional week ofculture. FIG. 1 represents an average of the results for both lines. Thegrowth rates of the lines during the two-week period followed a similarpattern (FIG. 2). Line LP3, however, metabolised more glucose and at theend of the 2-week period no glucose was detectable in the medium. Thissame line produced the largest amount of tissue (1278.18 g) at the endof the 4 week-liquid period when sucrose was used as sole carbon source.

[0062] After transferring the tissue to the prematuration medium Icontaining lactose, both lines maintained a similar growth pattern (FIG.3). During the second week in lactose, at the prematuration II stage,both lines showed a significant increase in the growth rates (FIG. 4).When media were analysed for the presence of glucose and galactose(FIGS. 5 and 6), detectable amounts of glucose and galactose wereobserved for both lines. A decrease of the amount of lactose was alsodetected. It is clear that both lines were capable of utilising lactose(Table 4). Data presented in FIG. 6 also provide grounds to concludethat the lines were not only capable of metabolising lactose but also ofutilising galactose, as a significant amount of “missing” galactose wasregistered at day 7 in comparison to day 5 (where the glucose amounts inthe medium were the same). TABLE 4 Sugar utilisation summary Amount ofsugar in media at Sugar the start, % % sugar missing (utilised) Sucrose1.0 30 Lactose 1.5 12

Example 5.1 Effect of Galactose During Proliferation and PrematurationSteps on Loblolly Pine

[0063] The finding that loblolly pine cultures were capable of utilisinggalactose is unique. In order to further investigate these phenomenontwo lines, LP1 and LP3, were cultured on semi-solid TX medium containing1.0% lactose and 0.5% sucrose. Cultures were maintained by subculturingthe embryogenic tissue every 10 to 14 days to fresh media. All cultureswere incubated at 23° C. in the dark. Suspension cultures were initiatedas described in Example 3, however, TX medium was supplemented witheither 1% sucrose, 1.5% maltose, 1.5% lactose, or 0.75% galactose. Thesecultures were maintained in liquid media for 4 weeks by weekly transfersto fresh maintenance or prematuration media. After the 4˜week period, 1ml of the liquid medium containing 0.1 g of tissue consisting of earlystage somatic embryos was plated on each 10 mm plates containing ½ LVmaturation medium. This medium contained 120 μM ABA and 6.0% sucrose and1% Phytagel. Eight to ten weeks later, mature somatic embryos werecounted and treatments were evaluated based on the number of somaticembryos per gram fresh weight (Table 5). TABLE 5 Effect of galactose andgalactose-containing sugars on SE yield Amount of tissue at the end ofthe Carbon source Line preculture II stage # SE/g FW Sucrose LP1 121.22 11 ± 3 LP3 1278.18 100 ± 6 Maltose LP1 232.18 106 ± 4 LP3 118.22 160 ±5 Lactose LP1 28.9 1060 ± 17 LP3 8.78 110 ± 7 Galactose LP1 59.45 216 ±8 LP3 8.65 172 ± 5

[0064] The results show that all lines were able to produce matureembryos after culturing in sucrose medium. However, they performeddifferently when maltose, lactose or galactose was used as a carbonsource. Line LP1 produced over 1000 embryos when cultured inlactose-containing TX medium and only 11 embryos when sucrose was usedas a carbon source. Although a reduction in embryo yield was observedwhen the same line was cultured in galactose-containing TX media, thenumber of embryos produced by LP1 when galactose was used as the carbonsource was significantly higher than the number obtained on sucrose- ormaltose-containing TX media. Line LP3 produced more embryos afterculturing in TX galactose-containing media. Again, the lowest embryoyield was observed when sucrose was used as carbon source. These resultssuggest that different lines require different types of sugar as acarbon source for their optimum performance, but galactose-containingsugars are superior.

[0065] Examples Assessing Radiata Pine Somatic Embryogenesis

Example 6 Effect of Carbon Source on Induction of Radiata Pine

[0066] Female gametophytes containing immature zygotic embryos were usedas explants. The cones were surface sterilised in 10% bleach solutionfor 15 min. They were rinsed 3 times with sterile water, air dried andstored in polyethylene bags at 4° C. until used. Seeds were sterilisedin 3% hydrogen peroxide containing 0.1% Tween 20 and rinsed in steriledeionized water. Megagametophytes containing the immature embryos wereplated on MSG (standard radiata pine medium containing sucrose, Table B)and on TX medium (Table A) supplemented with 2.2 mg/l 2,4-D, 1 mg/l BAand 0.3% Phytagel and containing 1.5% galactose and 0.5% sucrose. Caseinhydrolysate was reduced to 1000 mg/l (TXR medium). TABLE 8 MSG basalsalts Amount per liter Basal salts Medium, mg Major KNO₃ 100 KH₂PO₄ 170MgSO₄.7H₂O 370 CaCl₂.2H₂O 440 KCl 745 Minor KI 0.83 H₃BO₃ 6.2 MnSO₄.H₂O16.9 ZnSO₄.7H₂O 8.6 Na₂MoO₄.2H₂O 0.25 CuSO₄.5H₂O 0.025 CoCl₂.6H₂O 0.025Iron FeSO₄.7H₂O 27.8 Na₂EDTA 37.3 Vitamins Thiamine-HCl 0.1Pyridoxine-HCl 0.1 Nicotinic acid 0.5

[0067] Cultures were maintained on the two media by subculturing theembryogenic tissue every 10 to 14 days to fresh media. All cultures wereincubated at 23° C. in the dark. Suspension cultures were initiated as 2g of tissue were introduced to 50 ml of TXR liquid medium containing1.0% sucrose. These cultures were maintained in liquid media for 40-48days by transfers to fresh maintenance medium every 10 to 14 days.

[0068] One ml of TXR medium containing 0.1 g tissue consisting of earlystage somatic embryos was plated on modified ½ LV maturation medium(Table C) in which glutamine was reduced to 500 mg/l and caseinhydrolysate to 1000 mg/l. In addition, 120 μM ABA, 6.0% sucrose and 1%Phytagel were added. Twelve to fourteen weeks later, mature somaticembryos were counted and treatments were evaluated based on the numberof somatic embryos per gram fresh weight.

[0069] The results of the induction are presented in Table 6. Inductionsuccess was greater than three times higher when galactose was used as acarbon source and subsequent yields of mature embryos from these tissueswere almost double. TABLE C 1/2 LV medium basal salts Amount per literBasal salts Medium, mg Major NH₄NO₃ 825.00 KNO₃ 950.00 KH₂PO₄ 170.00MgSO₄.7H₂O 925.00 CaCl₂.2H₂O 11.00 Minor KI 4.1500 H₃BO₃ 31.0000ZnSO₄.7H₂O 43.0000 MnSO₄.H₂O 21.0000 Na₂MoO₄.2H₂O 1.3000 CuSO₄.5H₂O0.5000 CoCl₂.6H₂O 0.1300 Iron FeSO₄.7H₂O 27.8 Na₂EDTA 37.2 VitaminsThiamine-HCl 0.1000 Pyridoxine-HCl 0.1000 Nicotinic acid 0.5000Myo-Inositol 100

[0070] TABLE 6 Effect of carbon source on radiata pine induction successEmbryo yield Treatment % induction % cryopreserved (#SE/gFW) MSG 10.03100 213 TXR 26.07 100 405

Example 7 Effect of Carbon Source on Somatic Embryo Development ofRadiata Pine Embryos in Liquid Cultures

[0071] Tissue induced on MSG containing sucrose was further maintainedon MSG sucrose medium, and tissue induced on TXR galactose wasmaintained on TXR medium containing galactose as described in Example 6.The tissue morphology of the lines induced on galactose was differentfrom that induced on sucrose. Tissue induced on galactose-containingmedium consisted of more early stage immature embryos and a limitedamount of suspensor type of surrounding tissue, whereas predominantlysuspensor type tissue and very few embryos were observed in the tissueinduced on sucrose. The lines used in this study were RP1, RP2, RP3, andRP4.

[0072] Suspension cultures were initiated as described in Example 6.These cultures were maintained in liquid media for 20-28 days bytransfers to fresh maintenance medium every 10 to 14 days. Up to fivefold increase of the tissue was estimated at the end of each 10 to 14day transfer.

[0073] In order to study the effect of galactose on embryo developmentafter the proliferation stage, 2 g tissue was further transferred to 250ml flasks, containing 50 ml of TXR prematuration medium I supplementedwith either 1.0% sucrose or 3.0% galactose. As with loblolly pinecultures, prematuration I requires reduction of hormones either byreducing the auxin and cytokinin concentrations to optimally about{fraction (1/20)} of the concentration of proliferation step, or byadding charcoal at about 0.01 to 1%, or a combination of both.

[0074] After the 10-14 day period, 1.5 g tissue was transferred to 250ml flasks containing 50 ml TXR prematuration medium II to which either1.0% sucrose or 3.0% galactose and 90 μM ABA were added.

[0075] Early stage somatic embryos were collected after 10 to 14 daysand matured on modified ½ LV maturation medium as described in Example6. Results are shown in Table 7. TABLE 7 Tissue and somatic embryoyield. Grams FW at the end of Carbon source proliferation stage (from1.5 g) # SE/g FW ± sd Sucrose 11.05 ± 2 213.3 ± 6  Galactose  9.5 ± 2405.3 ± 10

[0076] Tissue growth increased more than 5 times when galactose was usedas a carbon source and the number of mature somatic embryos at the endof maturation was doubled.

[0077] Examples Assessing Douglas Fir Somatic Embryogenesis

[0078] The culture medium for Douglas fir was TX medium (see Table A)with the following additives: glutamine 100 mg., Casein hydrolysate 100mg., and pH 5.8. The following compounds were added into the basalmedium for Douglas fir somatic embryo development at different stages:

[0079] 1) Induction medium: 18 μM 2,4-D and 9 μM BA, phytagel 0.28%,sucrose 1%

[0080] 2) Solid maintenance medium: 9 μM 2,4-D and 4.5 μM BA, phytagel0.28%, sucrose 1%.

[0081] 3) Liquid maintenance and bulking-up medium: 9 μM 2,4-D and 4.5μM BA, different carbon sources for experiments.

[0082] 4) Liquid pre-treatment medium: 20 (first week) or 30 μM ABA(second week), 10% PEG4000 (first week) or PEG1500 (second week),different carbon sources as described in examples below.

[0083] 5) Solid maturation media: TX (Table A) plus 0.02% NH₄NO₃

[0084] Medium I. 40 μM ABA, 10% PEG 1500, maltose 2.5%, phytagel 0.6%

[0085] Medium II. 60 μM ABA, 10% PEG1500, maltose 2.5% and sucrose 1%,phytagel 0.8%

[0086] Medium III. 70 μM ABA, 6% PEG1500, sucrose 6%, phytagel 1%.

[0087] Embryogenic tissue was induced from immature zygotic embryos. Thecones and seeds were surface-sterilised as described for pines, andembryos were dissected out from megagametophytes. The embryos, with theattachment of suspensors to their megagametophytes, were placed on thesurface of induction medium. The culture was kept in darkness at 23° C.Embryogenic tissue was sub-cultured once every two weeks onto solidmaintenance medium.

Example 8 Effects of Carbohydrates on Liquid Maintenance of Douglas FirEmbryogenic Tissue

[0088] Embryogenic tissue of Douglas fir was collected from solidmaintenance culture and one gram of tissue was transferred into a 200 mlflask containing 50 ml TX (see Table A) liquid maintenance medium. Theliquid maintenance medium was supplied with different sugars (Table 8).TABLE 8 Growth of Douglas fir tissue (fold, mean ± SD) in suspensionmaintenance/bulking-up cultures for 3 weeks. Cell line DF-2 was used inthis experiment. Major carbon sources Additional glucose (%) Tissuegrowth 0.5% galactose 0 68.64 ± 4.58  0.5% galactose 0.025 97.02 ± 2.67   1% lactose 0 1.40 ± 0.14   1% lactose 0.025 2.75 ± 0.24 0.5% mannitol0 1.60 ± 0.13 0.5% mannitol 0.025 3.34 ± 0.22 0.5% glucose 0 223.14 ±15.4    1% sucrose 0 100.94 ± 12.0    1% maltose 0 85.01 ± 1.71 

[0089] The tissue was drained, weighed and sub-cultured once per week.During a culture period of three weeks, the highest tissue growth wasfound in the culture with glucose as the major carbon source. The mediacontaining sucrose, maltose and galactose also supported a fast tissuegrowth. With these latter sugars, tissue increased to 68 to 100 fold inthree weeks. Tissue grew little in the media containing lactose ormannitol. Supplement of a small amount of glucose in the mediacontaining galactose, lactose or mannitol respectively increased tissuegrowth (Table 8) and this increase of tissue growth corresponded to theamount of glucose supplied into the media (data not shown). Similarresults were obtained within two cell lines, DF-7A and DF-2.

[0090] Thus, among the tested sugars, glucose resulted in the fastesttissue growth. Sucrose, maltose and galactose were all good carbonsources for tissue growth. Lactose and mannitol have to be used incombination with other carbon sources such as glucose, in order toachieve a good tissue growth.

Example 9 Osmolarity and Carbon Metabolism in Liquid Maintenance

[0091] After a one-week liquid maintenance culture, the osmolarity ofculture medium showed little change when lactose or mannitol was used asthe major carbon source while the medium containing either galactose orglucose decreased from 22% to 32% (Table 9). More change of mediumosmolarity occurred during week 2 than week 1. The medium osmolaritychanged little when lactose or mannitol was used in the media. TABLE 9Medium osmolarity (mmol/kg, mean ± SD) and changes before and after aone-week tissue inoculation. Osmolarity before Osmolarity afterCarbohydrates inoculation inoculation Change (%) 0.5% galactose 71.67 ±1.15 48.67 ± 0.58 −32.09   1% lactose 93.33 ± 0.58 94.67 ± 1.15 1.440.5% mannitol 76 ± 1 71.67 ± 1.15 −5.70 0.5% glucose 77.67 ± 0.58 54.67± 1.53 −29.61

[0092] Sugars in the media were quantified with the method of enzymaticBioAnalysis that can quantify the specific carbohydrates in traceamount. In the medium, glucose decreased sharply, up to 94.4%, whilegalactose decreased about 80.6%. Little changes were found with lactoseor mannitol after a one-week culture (Table 10). TABLE 10 Changes ofcarbohydrates in Douglas fir suspension media after tissue (DF-7A),inoculation for one week. Major carbon Sugar Before use source analyzed(%) After use (%) Decreased (%) 0.5% glucose Glucose 0.5 0.0278 94.440.5% galactose Galactose 0.5 0.097 80.6   1% lactose Lactose 1 0.99 0.010.5% mannitol Mannitol 0.5 0.5 0

[0093] The results of this study show that glucose is aneasily-consumable carbon source for Douglas fir tissue. Galactose can beutilised slowly. Lactose and mannitol are not metabolised by Douglas firtissue.

Example 10 Effects of Carbohydrates in Suspension Cultures on EmbryoYield

[0094] In pre-treatment media, ABA was the only plant hormone present inthe media and polyethylene glycol (PEG) was added as a non-permeableosmoticum to stimulate and synchronize embryo maturation. It was acommon phenomenon that tissue grew more after being transferred frommaintenance media containing other carbon sources into the pre-treatmentmedium containing maltose as the major carbon source. Tissue grew fasterduring the first week and then slowed down in the second week (Table11).

[0095] After pre-treatment, about 0.1 g tissue was plated onto a pieceof filter paper on the surface of solid maturation medium I. After twoweeks, the tissue was transferred with the filter paper to medium II andthen medium III after three additional weeks. The maturation culture waskept in darkness, 23° C. for 8 weeks and then 12° C. for one week.Embryos were evaluated after this nine-week maturation. Duringevaluation only mature embryos with a normal shape and at least threewell-developed cotyledons were scored. TABLE 11 Douglas fir tissuegrowth (fold, mean ± SD) during pre-maturation treatments (pt) andembryo maturation. Yield of Carbon source embryos (started inmaintenance Growth - Pt Growth - Pt Total Mature with one gram of &pre-treatment week 1 week 2 growth embryos/pp tissue at P1) A* & A  4.9± 0.28 2.4 ± 0.28 11.76   75 ± 23.3 8017 A & m**  7.1 ± 0.85 2.3 ± 0.1416.33   105 ± 27.81 15586 B & B 4.75 ± 1.06 1.9 ± 0.28 9.025 61.38 ±19.64 5035 B & m  7.7 ± 0.74 2.4 ± 0.6  18.48 82.33 ± 12.5  13830 C & C6.85 ± 0.64  2 ± 0.8 13.7   53 ± 25.23 6600 C & m  8.5 ± 0.62 2.2 ± 0.4 18.7 96.75 ± 20.65 16446 D & D  9.9 ± 0.57 2.3 ± 0.21 22.77   62 ± 15.412834 D & m 9.4 ± 0.6 2.4 ± 0   22.56 105.75 ± 20.36  21686 Suc & m 6.6± 0.2 4.92 ± 0.28  32.472  50.5 ± 19.27 14906

[0096] Galactose and a small amount of glucose supplement in liquidmaintenance culture combined with maltose in pre-treatment showed thehighest embryo yield either in the total number or on the yield per gramof tissue.

1. A method for reproducing conifers by somatic embryogenesis wherein agalactose-containing compound is used as a carbon source for anembryogenic culture during at least one of the steps of induction,proliferation, and prematuration.
 2. The method of claim 1 wherein thegalactose-containing compound is a sugar with galactose comprising oneor more of the sub-units.
 3. The method of claim 2 wherein thegalactose-containing sugar is selected from the group consisting ofmonosaccharides, disaccharides, oligosaccharides, and polysaccharides.4. The method of claim 3 wherein the galactose-containing sugar islactose.
 5. The method of claim 1 wherein the galactose-containingcompound is less than about 6% of the nutrient medium.
 6. The method ofclaim 1 wherein the nutrient medium is gelled or liquid.
 7. The methodof claim 1 wherein the conifers are selected from the family Pinaceae.8. The method of claim 7 wherein the conifers are selected from thegenera Pinus, Picea and Pseudotsuga.
 9. The method of claim 8 whereinthe conifer is Pinus taeda or a hybrid thereof.
 10. The method of claim8 wherein the conifer is Pseudotsuga menziesii.
 11. The method of claim8 wherein the conifer is Pinus radiata.
 12. The method of claim 1 inwhich the embryogenic culture is cultured in at least one prematurationmedium comprising a galactose-containing compound and then transferredto a maturation medium to produce cotyledonary stage embryos suitablefor germination.
 13. The method of claim 12 wherein the prematurationmedium contains less auxin and less cytokinin than the nutrient mediumused during proliferation.
 14. The method of claim 12 wherein theprematuration medium further comprises abscisic acid.
 15. The method ofclaim 3 wherein the galactose-containing sugar is supplemented withadditional sugars.
 16. The method of claim 15, wherein the additionalsugars are readily metabolized.
 17. The method of claim 16, wherein theadditional sugars are selected from the group consisting of sucrose,glucose, and fructose.
 18. The method of claim 1 wherein thegalactose-containing compound is more than about 1% of the nutrientmedium.
 19. The method of claim 1 wherein the embryogenic culturecontains early stage embryos.
 20. The method of claim 1 wherein thegalactose-containing compound is less than about 2% of the nutrientmedium.
 21. The method of claim 1 wherein the galactose-containingcompound is between about 1% and about 6% of the nutrient medium. 22.The method of claim 1 wherein the nutrient medium further comprises anauxin and a cytokinin.
 23. A method for reproduction by somaticembryogenesis of conifers selected from the group consisting of Pinustaeda and hybrids, Pinus radiata, and Pseudotsuga menziesii whichcomprises: using a galactose-containing compound during at least one ofthe steps of induction, proliferation, and prematuration.
 24. The methodof claim 23 wherein the galactose-containing compound is a sugar withgalactose comprising one or more of the sub-units.
 25. The method ofclaim 24 wherein the galactose-containing sugar is selected from thegroup consisting of monosaccharides, disaccharides, oligosaccharides,and polysaccharides.
 26. The method of claim 25 wherein thegalactose-containing sugar is lactose.
 27. The method of claim 23wherein the galactose-containing compound is less than about 6% of thenutrient medium.
 28. The method of claim 23 wherein the nutrient mediumis gelled or liquid.
 29. The method of claim 23 wherein the conifer isPinus taeda or a hybrid thereof.
 30. The method of claim 23 wherein theconifer is Pseudotsuga menziesii.
 31. The method of claim 23 wherein theconifer is Pinus radiata.
 32. The method of claim 23 in which theembryogenic culture is cultured in at least one prematuration mediumcomprising a galactose-containing compound and then transferred to amaturation medium to produce cotyledonary stage embryos suitable forgermination.
 33. The method of claim 32 wherein the prematuration mediumcontains less auxin and less cytokinin than the nutrient medium usedduring proliferation.
 34. The method of claim 32 wherein theprematuration medium further comprises abscisic acid.
 35. The method ofclaim 24 wherein the galactose-containing sugar is supplemented withadditional sugars.
 36. The method of claim 35, wherein the additionalsugars are readily metabolized.
 37. The method of claim 36, wherein theadditional sugars are selected from the group consisting of sucrose,glucose, and fructose.
 38. The method of claim 23 wherein thegalactose-containing compound is more than about 1% of the nutrientmedium.
 39. The method of claim 23 wherein the embryogenic culturecontains early stage embryos.
 40. The method of claim 23 wherein thenutrient medium further comprises an auxin and a cytokinin.
 41. Themethod of claim 23 wherein the galactose-containing compound is lessthan about 2% of the nutrient medium.
 42. The method of claim 23 whereinthe galactose-containing compound is between about 1% and about 6% ofthe nutrient medium.
 43. A method for reproducing conifers by somaticembryogenesis which comprises: growing conifer cells on a nutrientmedium comprising a galactose-containing compound, an auxin, and acytokinin to produce an embryogenic culture.